Fluorescence/Förster Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) both allow real-time detection of protein-protein interactions in intact cells. FRET involves energy transfer between two fluorophores (fluorescent proteins). A donor fluorophore, initially in its electronic excited state, may transfer energy to an acceptor fluorophore through nonradiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to small distances. Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other. A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise (autofluorescence, light scattering and/or photoisomerization) due to direct excitation of the acceptor or to photobleaching. Photobleaching is an important drawback when assessing endogenous interactions because it can damage the cells and therefore alter normal interactions between biomolecules.
To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. BRET assay technology is based on the efficient resonance energy transfer (RET) between a bioluminescent donor moiety and a fluorescent acceptor moiety. This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than a fluorophore (typically Cyan fluorescent protein (CFP)) to produce an initial photon emission compatible with the fluorescent acceptor (typically yellow fluorescent protein (YFP)).
FRET allows reliable monitoring of sequential transfer between three fluorophores in a three-color or triple-FRET assay, but still suffers from photobleaching of the donor and contaminating cross-excitation problems. Sequential Resonance Energy Transfer (SRET), which combines serially BRET between an initial donor and intermediate acceptor and resonance energy transfer between the intermediate acceptor and a terminal acceptor (FIG. 1B), is based on the same principle using bioluminescence as a source of energy, but is handicapped by a low signal output and high signal cross-contamination. Coupling a protein complementation assay (PCA) to BRET or FRET can also detect ternary complexes. However, PCA imposes greater steric constraints for permissive interactions, slow dissociation of interacting partners due to stable folding of the reconstituted donor and also suffers from low output signal intensity.
As many biomolecules interact in ternary (or higher order complexes), there remains a need for assays that allow for reliable ternary/quaternary complex monitoring.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.